Distribution and characterization of bacterial communities in diverse Antarctic ecosystems by high-troughput sequencing

Geological events and historical climate changes have eliminated or reduced most life in Antarctica to mainly microbial organisms in relatively simple communities. Due to its exceptional location, millennia long isolations and extreme climatic conditions, the continent offers a spectacular and unique background for fundamental scientific research and the testing of hypotheses. Notwithstanding the fact that Antarctica is still considered by many to be one of the last pristine environments on Earth, it is not only threatened by climate change, which particularly has severe effects on parts of West and Maritime Antarctica, but also by an ever increasing number of tourists and even scientists themselves. Studies on Antarctic biota are relatively scarce, and despite the fact that bacteria are fundamental to the Antarctic ecosystems, only a minority of the studies focus on these organisms. This results in a lacuna in the knowledge about the diversity, distribution and functioning of and the relationships between these organisms under the extreme Antarctic conditions. The recent advent of High-throughput sequencing (HTS) applications enables to sequence millions of DNA-fragments in a very short time, allowing us to visualise bacterial communities at a very high resolution, without the necessity for prior isolation of the organisms. In this PhD-study, we have applied some of these new technologies in order to investigate the bacterial diversity of different habitats throughout the Antarctic. In a first study (Chapter 2), we have compared the results obtained by pyrosequencing and compared these with the results of a previous isolation campaign. As expected, a much larger diversity of bacteria were found with pyrosequencing. While five bacterial phyla were recovered by cultivation, this was the case for 22 phyla with the NGS-approach, and a large amount of unknown diversity was evident. At the same time, it became clear that also the part of the 16S rRNA gene that was sequenced had an impact on the perceived diversity, with the V1-V2 fragments resulting in ~50% more OTUs than the V3-V2 fragments and only a limited amount of overlap in the genera recovered was noticed. In contrast, more chimeric sequences were identified in the V3-V2 amplicons. Notwithstanding the fact that pyrosequencing yielded a higher diversity, there was very little overlap with the cultivation approach, with only about 4 % of the OTUs recovered by cultivation found with pyrosequencing. In contrast, we also noticed that some singleton pyrosequencing OTUs where easily grown on growth media, and hence were not errors in the pyrosequencing data. This study thus showed that several factors could have a large impact on the perceived diversity, and that complementary techniques are necessary to discover the total bacterial diversity. In a second study (Chapter 3), we have examined the effects of both different bedrock types (granite and gneiss) and the presence of macrobiota (mosses, lichens and algae) on the composition of bacterial communities in high-altitude inland soils of different regions if the western Sør Rondane Mountains (Queen Maud Land, East Antarctica), near the Belgian Princess Elisabeth research station. We have used the at present most used HTS-platform, Illumina’s MiSeq, which allows sequencing longer gene fragments and yields more sequences compared to pyrosequencing. We combined this with the ARISA genetic fingerprinting technique. We demonstrated that organic carbon was the most significant parameter in structuring bacterial communities, followed by pH, electric conductivity, bedrock type and moisture content, while spatial distance was of less importance. Diversity showed a positive correlation with moisture content. Acidobacteria and Actinobacteria dominated dry gneiss derived mineral soils, while Proteobacteria, Cyanobacteria, Armatimonadetes and candidate phylum FBP were dominant in samples with a high organic carbon content. A large part of the unexplained variation is probably caused by the absence of data about important nutrients in our dataset (nitrogen and phosphorous), together with microclimatic and topographic differences between sample locations, and noise and stochasticity. In a last study we again used the Illumina MiSeq platform to perform a pan-continental charting of benthic and littoral microbial mats. In total, 138 samples from lakes in eight Antarctic regions and two Sub-Antarctic islands were analysed. We found a significant trend of increasing biodiversity with decreasing latitude from 85° to 54° S, although this than again decreased until 45° S. The mean annual temperature appeared to have a highly significant effect on community structure between Sub-Antarctica and Antarctica, while, besides the geographical distance, electric conductivity, and to a lesser extent pH, was important in explaining differences between samples on the Antarctic continent. In this study, too, a very high unknown diversity was observed. Particularly Cyanobacteria and Alphaproteobacteria dominated freshwater microbial mats, while Bacteroidetes and the alphaproteobacterial Rhodobacteraceae family dominated saline lakes. The Sub-Antarctic Marion Island was highly deviant with very low species richness, dominated by Janthinobacterium (Betaproteobacteria). In conclusion this thesis supports the hypothesis that for Bacteria in the Antarctic Region, too, distinct biogeographic patterns exist and that the environment exerts large selective pressures on community structure and composition, complemented by biotic factors. There is a high amount of heterogeneity at both local and continental scale due to both spatial distance and local differences in environmental variables such as electric conductivity, pH, moisture content, organic carbon and microclimate. Although we only were able sample a fraction of the continent, it is expected that similar patterns hold across the entire continent. However, additional sampling and in depth (metagenomics) sequencing linked to extensive environmental data, combined with phylogenetic analysis is needed to resolve important questions such as within and inter-continental dispersal, functioning and correlation of observed patterns to environmental data

Highly diverse nirK genes comprise two major clades that harbour ammonium-producing denitrifiers

Background: Copper dependent nitrite reductase, NirK, catalyses the key step in denitrification, i.e. nitrite reduction to nitric oxide. Distinct structural NirK classes and phylogenetic clades of NirK-type denitrifiers have previously been observed based on a limited set of NirK sequences, however, their environmental distribution or ecological strategies are currently unknown. In addition, environmental nirK-type denitrifiers are currently underestimated in PCR-dependent surveys due to primer coverage limitations that can be attributed to their broad taxonomic diversity and enormous nirK sequence divergence. Therefore, we revisited reported analyses on partial NirK sequences using a taxonomically diverse, full-length NirK sequence dataset. Results: Division of NirK sequences into two phylogenetically distinct clades was confirmed, with Clade I mainly comprising Alphaproteobacteria (plus some Gamma- and Betaproteobacteria) and Clade II harbouring more diverse taxonomic groups like Archaea, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Nitrospirae, Firmicutes, Actinobacteria, Planctomycetes and Proteobacteria (mainly Beta and Gamma). Failure of currently available primer sets to target diverse NirK-type denitrifiers in environmental surveys could be attributed to mismatches over the whole length of the primer binding regions including the 3' site, with Clade II sequences containing higher sequence divergence than Clade I sequences. Simultaneous presence of both the denitrification and DNRA pathway could be observed in 67 % of all NirK-type denitrifiers. Conclusion: The previously reported division of NirK into two distinct phylogenetic clades was confirmed using a taxonomically diverse set of full-length NirK sequences. Enormous sequence divergence of nirK gene sequences, probably due to variable nirK evolutionary trajectories, will remain an issue for covering diverse NirK-type denitrifiers in amplicon-based environmental surveys. The potential of a single organism to partition nitrate to either denitrification or dissimilatory nitrate reduction to ammonium appeared to be more widespread than originally anticipated as more than half of all NirK-type denitrifiers were shown to contain both pathways in their genome

Analysis of cbbL, nifH, and pufLM in soils from the Sør Rondane Mountains, Antarctica, reveals a large diversity of autotrophic and phototrophic bacteria

Cyanobacteria are generally thought to be responsible for primary production and nitrogen fixation in the microbial communities that dominate Antarctic ecosystems. Recent studies of bacterial communities in terrestrial Antarctica, however, have shown that Cyanobacteria are sometimes only scarcely present, suggesting that other bacteria presumably take over their role as primary producers and diazotrophs. The diversity of key genes in these processes was studied in surface samples from the Sor Rondane Mountains, Dronning Maud Land, using clone libraries of the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL, cbbM) and dinitrogenase-reductase (nifH) genes. We recovered a large diversity of non-cyanobacterial cbbL type IC in addition to cyanobacterial type IB, suggesting that non-cyanobacterial autotrophs may contribute to primary production. The nifH diversity recovered was predominantly related to Cyanobacteria, particularly members of the Nostocales. We also investigated the occurrence of proteorhodopsin and anoxygenic phototrophy as mechanisms for non-Cyanobacteria to exploit solar energy. While proteorhodopsin genes were not detected, a large diversity of genes coding for the light and medium subunits of the type 2 phototrophic reaction center (pufLM) was observed, suggesting for the first time, that the aerobic photoheterotrophic lifestyle may be important in oligotrophic high-altitude ice-free terrestrial Antarctic habitats

Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats : a comparison between bidirectional pyrosequencing and cultivation

The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1–22.2%) and OTU (3.5–3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1–V2 and V3–V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity

Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments

High‐throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species‐level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum‐specific species‐level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read‐merging and two clustering algorithms and the cluster‐free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus‐level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species‐level detections, which were, however, associated with higher error rates compared to ASVs. Genus‐level assignments using ASVs exhibited higher accuracy and lower error rates compared to species‐level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples

MICROBIAN : Microbial diversity in the Sør Rondane Mountains in a context of climate change

The Sør Rondane Mountains (SRM) represent a c. 900 km² large mountain range, encompassing a large range of terrestrial habitats differing in geology and soil characteristics, exposure time and microclimatic conditions. The objectives of the BelSPO project MICROBIAN are to (i) use a combination of remote sensing (Digital Elevation Model) and close-range field observation techniques to map physical habitat characteristics and the presence/extent of biological crust communities in the region of the Princess Elisabeth Station Antarctica (PEA), (ii) generate a comprehensive inventory of the taxonomic and functional diversity of microbial communities in these habitats by amplicon sequencing of the 16S and 18S rRNA genes and metagenomics, (iii) use mesocosm field experiments (Open Top Chambers and snow fences) to mimic the possible effects of future climate change on the taxonomic diversity of these microbial ecosystems, and (iv) conduct field experiments to inform policy-makers in view of decision making regarding environmental protection and prevention measures to reduce the introduction and spread of non-native species and to avoid cross-contamination between sites. The proposed research will provide a proof of concept to use high resolution satellite images for identifying regions of particular biological interest in East Antarctica and more broadly make a significant contribution to understanding Antarctic terrestrial microbial ecology.MICROBIA

The science-policy link in practice: how to propose an Antarctic Specially Protected Area (ASPA)?

The BelSPO projects ANTAR-IMPACT, BELDIVA and MICROBIAN and literature data concerning the biodiversity in the inland biotopes of the Western Sør Rondane Mountains (Dronning Maud Land) indicated a very rich and unique terrestrial biodiversity on the nunataks in the surroundings of Princess Elisabeth Station, including biofilms and Biological Soil Crusts. However, there is potential for negative impacts due to visits and (human) disturbances, mainly driven by the presence of infrastructures in the vicinity. Therefore, the involved scientists initiated the process of creating an ASPA in collaboration with the relevant ministries (Foreign Affairs, Environment and Science Policy). The first step was the submission of a Working Paper (WP42) at CEP XX (2017) with a Preliminary assessment of the values to be protected, using the template of Appendix 4 to the CEP XX report. Other countries scrutinized the document and raised useful comments and questions. The Information Paper (IP42) submitted at CEP XXI (2018) contained the answers to these questions. The next step was the writing of a Management Plan, based on a Guide that includes all the elements to describe (annex to Resolution 2 (2011)). Several versions were discussed, also with the station operator. The WP15 was finally presented to CEP XXIV that forwarded it to the Subsidiary Group on Management Plan, where it will be further improved during the intersessional period. The process was a learning experience for the scientists, from creating the maps to developing management options, and lastly communicating the importance of the area to policy- and decision-makers.MICROBIAN15. Life on lan

Polar lake microbiomes have distinct evolutionary histories

Toward the poles, life on land is increasingly dominated by microorganisms, yet the evolutionary origin of polar microbiomes remains poorly understood. Here, we use metabarcoding of Arctic, sub-Antarctic, and Antarctic lacustrine benthic microbial communities to test the hypothesis that high-latitude microbiomes are recruited from a globally dispersing species pool through environmental selection. We demonstrate that taxonomic overlap between the regions is limited within most phyla, even at higher-order taxonomic levels, with unique deep-branching phylogenetic clades being present in each region. We show that local and regional taxon richness and net diversification rate of regionally restricted taxa differ substantially between polar regions in both microeukaryotic and bacterial biota. This suggests that long-term evolutionary divergence resulting from low interhemispheric dispersal and diversification in isolation has been a prominent process shaping present-day polar lake microbiomes. Our findings illuminate the distinctive biogeography of polar lake ecosystems and underscore that conservation efforts should include their unique microbiota

Long-term N-addition alters the community structure of functionally important N-cycling soil microorganisms across global grasslands

Anthropogenic nitrogen (N) input is known to alter the soil microbiome, but how N enrichment influences the abundance, alpha-diversity and community structure of N-cycling functional microbial communities in grasslands remains poorly understood. Here, we collected soils from plant communities subjected to up to 9 years of annual N-addition (10 g N m−2 per year using urea as a N-source) and from unfertilized plots (control) in 30 grasslands worldwide spanning a large range of climatic and soil conditions. We focused on three key microbial groups responsible for two essential processes of the global N cycle: N2 fixation (soil diazotrophs) and nitrification (AOA: ammonia-oxidizing archaea and AOB: ammonia-oxidizing bacteria). We targeted soil diazotrophs, AOA and AOB using Illumina MiSeq sequencing and measured the abundance (gene copy numbers) using quantitative PCR. N-addition shifted the structure of the diazotrophic communities, although their alpha-diversity and abundance were not affected. AOA and AOB responded differently to N-addition. The abundance and alpha-diversity of AOB increased, and their community structure shifted with N-addition. In contrast, AOA were not affected by N-addition. AOA abundance outnumbered AOB in control plots under conditions of low N availability, whereas N-addition favoured copiotrophic AOB. Overall, N-addition showed a low impact on soil diazotrophs and AOA while effects for AOB communities were considerable. These results reveal that long-term N-addition has important ecological implications for key microbial groups involved in two critical soil N-cycling processes. Increased AOB abundance and community shifts following N-addition may change soil N-cycling, as larger population sizes may promote higher rates of ammonia oxidation and subsequently increase N loss via gaseous and soil N-leaching. These findings bring us a step closer to predicting the responses and feedbacks of microbial-mediated N-cycling processes to long-term anthropogenic N-addition in grasslands

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

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